Mark Battle

Monitoring Tumor Response to Antiangiogenic Sunitinib Therapy with 18F-Fluciclatide, an 18F-Labeled αVβ3-Integrin and αVβ5-Integrin Imaging Agent

Arginine-glycine-aspartate (RGD)-binding αVβ3-integrin and αVβ5-integrin play key roles in tumor angiogenesis. We examined an 18F-labeled small peptide (fluciclatide [United States Adopted Name (ASAN)–approved, International Nonproprietary Name (INN)–proposed name], previously referred to as AH111585) containing an RGD sequence. Fluciclatide binds with a high (nM) affinity to αVβ3-integrin and αVβ5-integrin, which are highly expressed on tumors and the tumor neovasculature. In this study, 18F-fluciclatide was used to examine the response of human glioblastoma xenografts to treatment with the antiangiogenic agent sunitinib. Methods: U87-MG tumor uptake of 18F-fluciclatide was determined by small-animal PET after longitudinal administration of the antiangiogenic agent sunitinib (a 2-wk dosing regimen). Tumor sizes were measured throughout the study, and tumor volumes were calculated. Tumor microvessel density (MVD) after therapy was also analyzed. Results: Dynamic small-animal PET of 18F-fluciclatide uptake after administration of the clinically relevant antiangiogenic agent sunitinib revealed a reduction in the tumor uptake of 18F-fluciclatide compared with that in vehicle-treated controls over the 2-wk dosing regimen. Skeletal muscle, used as a reference tissue, showed equivalent 18F-fluciclatide uptake in both therapy and control groups. A reduction in tumor MVD was also observed after treatment with the antiangiogenic agent. No significant changes in tumor volume were observed in the 2 groups. Conclusion: The data demonstrated that 18F-fluciclatide detected changes in tumor uptake after acute antiangiogenic therapy markedly earlier than any significant volumetric changes were observable. These results suggest that this imaging agent may provide clinically important information for guiding patient care and monitoring the response to antiangiogenic therapy.

Use of 2-[18F]fluoroethylazide for the Staudinger ligation – Preparation and characterisation of GABAA receptor binding 4-quinolones

The labelling reagent 2-[(18)F]fluoroethylazide was used in a traceless Staudinger ligation. This reaction was employed to obtain the GABA(A) receptor binding 6-benzyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (2-[(18)F]fluoroethyl) amide. The radiotracer was prepared with a non-decay corrected radiochemical yield of 7%, a radiochemical purity >95% and a specific radioactivity of 0.9 GBq/micromol. The compound showed low brain penetration in normal rats. A series of fluoroalkyl 4-quinolone analogues with nanomolar to sub-nanomolar affinity for the GABA(A) receptor has been prepared as well.

99mTc-NC100692--a tracer for imaging vitronectin receptors associated with angiogenesis: a preclinical investigation.

INTRODUCTION Technetium 99m (99mTc)-NC100692 is being developed as a marker of vitronectin receptor expression. The purpose of this study was to confirm the binding affinity [dissociation constant (Kd)] of 99mTc-NC100692 for a range of integrin receptors including alphavbeta3 and alphavbeta5 as well as to establish the biodistribution and metabolic stability of 99mTc-NC100692 in Wistar rats. METHODS The Kd of 99mTc-NC100692 for a range of human integrin receptors was established in an in vitro saturation binding assay. The biodistribution and metabolic stability of 99mTc-NC100692 in normal Wistar rats was investigated. RESULTS The Kd of 99mTc-NC100692 to alphavbeta3 and alphavbeta5 was less than 1 nM. It was not possible to saturate the binding of 99mTc-NC10092 towards alphaIIbbeta3, alpha5beta1, alpha3beta1 or alpha1beta1, and as a result, accurate Kd values could not be determined. The biodistribution of 99mTc-NC100692 in male and female Wistar rats showed that radioactivity was rapidly excreted, predominantly into the urine, with very little background tissue retention apart from the liver and kidneys. Kidney and liver retention was reduced in the presence of excess NC100692 ligand. In vivo, there was little systemic metabolism of 99mTc-NC100692. CONCLUSIONS 99mTc-NC100692 has a high affinity for the vitronectin receptors that are associated with angiogenesis. 99mTc-NC100692 is metabolically stable in the systemic circulation of rats with a biodistribution that is favourable for imaging purposes. This evidence suggests that 99mTc-NC100692 might be a useful marker of vitronectin receptor expression in vivo.

The development of potential new fluorine-18 labelled radiotracers for imaging the GABAA receptor

Positron emission tomography (PET) using the tracer [(11)C]Flumazenil has shown changes in the distribution and expression of the GABA(A) receptor in a range of neurological conditions and injury states. We aim to develop a fluorine-18 labelled PET agent with comparable properties to [(11)C]Flumazenil. In this study we make a direct comparison between the currently known fluorine-18 labelled GABA(A) radiotracers and novel imidazobenzodiazepine ligands. A focussed library of novel compound was designed and synthesised where the fluorine containing moiety and the position of attachment is varied. The in vitro affinity of twenty-two compounds for the GABA(A) receptor was measured. Compounds containing a fluoroalkyl amide or a longer chain ester group were eliminated due to low potency. The fluorine-18 radiochemistry of one compound from each structural type was assessed to confirm that an automated radiosynthesis in good yield was feasible. Eleven of the novel compounds assessed appeared suitable for in vivo assessment as PET tracers.

c-Met PET Imaging Detects Early-Stage Locoregional Recurrence of Basal-Like Breast Cancer

Locoregional recurrence of breast cancer poses significant clinical problems because of frequent inoperability once the chest wall is involved. Early detection of recurrence by molecular imaging agents against therapeutically targetable receptors, such as c-Met, would be of potential benefit. The aim of this study was to assess 18F-AH113804, a peptide-based molecular imaging agent with high affinity for human c-Met, for the detection of early-stage locoregional recurrence in a human basal-like breast cancer model, HCC1954. Methods: HCC1954 tumor–bearing xenograft models were established, and 18F-AH113804 was administered. Distribution of radioactivity was determined via PET at 60 min after radiotracer injection. PET and CT images were acquired 10 d after tumor inoculation, to establish baseline distribution and uptake, and then on selected days after surgical tumor resection. CT images and caliper were used to determine the tumor volume. Radiotracer uptake was assessed by 18F-AH113804 PET imaging. c-Met expression was assessed by immunofluorescence imaging of tumor samples and correlated with 18F-AH113804 PET imaging results. Results: Baseline uptake of 18F-AH113804, determined in tumor-bearing animals after 10 d, was approximately 2-fold higher in the tumor than in muscle tissue or the contralateral mammary fat pad. The tumor growth rate, determined from CT images, was comparable between the animals with recurrent tumors, with detection of tumors of low volume (<10 mm3) only possible by day 20 after tumor resection. 18F-AH113804 PET detected local tumor recurrence as early as 6 d after surgery in the recurrent tumor–bearing animals and exhibited significantly higher 18F-AH113804 uptake (in comparison to mammary fatty tissue), with a target-to-background (muscle) ratio of approximately 3:1 (P < 0.01). The c-Met expression of individual resected tumor samples, determined by immunofluorescence, correlated with the respective 18F-AH113804 imaging signals (r = 0.82, P < 0.05). Conclusion: 18F-AH113804 PET provides a new diagnostic tool for the detection of c-Met–expressing primary tumor and has potential utility for the detection of locoregional recurrence from an early stage.

Evaluation of a novel series of fluorine-18-labeled imidazobenzodiazepines as potential new positron emission tomography radioligands for the GABAA receptor.

INTRODUCTION [(11)C]Flumazenil has been used to study the GABAA receptor in many preclinical and clinical studies, but the short half-life of carbon-11 means that this molecule is restricted to use by investigators with access to on-site cyclotron and radiosynthesis facilities. The radiosynthesis of [(18)F]flumazenil has been evaluated by several groups, but the radiochemical yield can be low and inconsistent. We previously reported a series of fluorine-18-labeled imidazobenzodiazepine-based ligands for the GABAA receptor, which had significantly improved radiosynthesis yields. Here we report the in vivo evaluation and comparison of the distribution, metabolism and specificity of the novel ligands in comparison with [(18)F]flumazenil. METHODS In vivo biodistribution studies, at time points up to 90min post-injection, were performed in naïve rats to compare the performance of the novel compounds with particular attention paid to regional brain uptake and clearance. In vivo metabolism studies were carried out to determine the percentage of parent compound remaining in the plasma and brain at selected time points. Blocking studies were carried out, using pre-treatment of the test animals with either bretazenil or unlabeled fluorine-19 test compound, to determine the levels of specific and non-specific binding in selected brain regions. RESULTS Two of the 12 new compounds were rejected due to poor biodistribution showing significant bone uptake. Some of the compounds showed insufficient whole brain uptake or limited evidence of differential binding to GABAA-rich brain regions to warrant further investigation. Four of the compounds were selected for in vivo metabolism and blocking studies. Overall, the studies indicated that two compounds 3 and 5 showed comparable or improved performance compared with [(18)F]flumazenil, with respect to distribution, metabolic profile and specific binding. CONCLUSIONS These studies have demonstrated that compounds based on [(18)F]flumazenil, but with alterations to allow improved radiosynthesis, can be prepared which have ideal properties and warrant further evaluation as PET agents for the GABAA receptor. In particular, compounds 3 and 5 show very promising profiles with specific binding and in vivo stability comparable to flumazenil.

Abstract 357: Nonclinical tumor efficacy studies of [18F]AH113804, a novel PET imaging agent with high affinity for the human c-Met receptor

c-Met, the tyrosine-kinase receptor for hepatocyte growth factor (HGF)/scatter factor, is involved in tumour growth, invasion and metastasis in many human cancers of epithelial origin. Various tumour types are reported to overexpress c-Met, whilst expression in most normal tissues is relatively low. [18F]AH113804 is a peptide-based molecular imaging agent being developed by GE Healthcare for the in vivo evaluation of tumour and metastatic c-Met expression by PET imaging. In vitro affinity studies with fluorescently labelled analogues confirmed that whilst the AH113804 peptide had high affinity for human c-Met (hc-Met), there was limited or no affinity for dog or rodent c-Met, respectively. In mice, relatively high uptake of [18F]AH113804 was observed in human xenograft tumours known to express high levels of hc-Met, with rapid clearance from key background tissues such as muscle (tumour to muscle ratio of >5 achieved by 30 minutes post-injection). This biodistribution profile allowed the tumour to be clearly visualised by micro-PET imaging. Tumour uptake was significantly reduced by co-administration of excess non-radioactive peptide, confirming tumour uptake was specific. Tumour uptake of [18F]AH113804 was also shown to correlate with expression of hcMet, with a relative retention of 2.0±0.1, 1.2±0.2 and 0.7±0.2% retained dose per gram normalised for body weight (% rd/g/100g bw) 60 minutes post-injection in xenograft tumours with relatively high (HT-29 tumours), lower (U87 tumours) and no (LLC tumours) expression of hc-Met respectively (as assayed by ELISA). Finally, 111InCl3 was used in a dual tumour model as a non-specific marker of blood pool to confirm differences in tumour uptake were not related to differences in tumour blood pool or delivery. There was a significant difference in [18F]AH113804 uptake between HT-29 (2.2±0.8% rd/g/100 g bw) and LLC (0.9±0.2% rd/g/100 g bw) tumours (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 357. doi:1538-7445.AM2012-357

The in vivo and in vitro metabolic profile of 99mTc-NC100668, a new tracer for imaging venous thromboembolism: Identification and biodistribution of the principal radiolabeled metabolite

99mTc-NC100668 [Acetyl-Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr(3-iodo)-Thr-Leu-Leu-Lys-Gly-NC100194] is a radiopharmaceutical imaging agent being developed to aid the diagnosis of thromboembolism. The stability profile of 99mTc-NC100668 was investigated by high-performance liquid chromatography (HPLC) after in vitro exposure to blood and plasma obtained from rat and human, as well as to urine and bile obtained from rat. The metabolic profile of 99mTc-NC100668 exposed to human and rat hepatic S9 (a liver homogenate-rich cytochrome P450) was also studied. The profile of 99mTc-labeled species in plasma, urine, and bile was investigated following i.v. administration of 99mTc-NC100668 to rat. The major species observed in vitro and in vivo consisted of the 99mTc-chelator (NC100194) [N,N-Bis(N-(1,1-dimethyl-2-(hydroxylimino-)propyl)aminoethyl)aminoethylamine] attached to the C-terminal amino acid residue and referred to as 99mTc-complex of Gly-NC100194. The identity of the major metabolite was confirmed by cochromatography with an authentic standard and the genuine metabolite using a second HPLC method. The minor metabolites were sodium pertechnetate (99mTc) and 99mTc-NC100194. In addition, a small number of other species were transiently observed in vitro; they were not investigated further. The biodistribution of the major metabolite was studied in male Wistar rats. The affinity of the major metabolite toward plasma clot was established using a plasma clot-forming assay. A minor uptake of 99mTc-complex of Gly-NC100194 in the plasma clot and a rapid removal from the body were noted. In conclusion, the metabolites of 99mTc-NC100668 are not anticipated to have a negative impact on the ability of the test substance to image blood clots.

UTILITY OF PMOD IMAGE QUANTIFICATION SOFTWARE FOR PROCESSING [11C]PIB AND [18F]FLUTEMETAMOL IMAGES FOR SUVR QUANTITATION ON THE CENTILOID SCALE

analysis. Results: In the early AD dementia group, we found a significantly increased DBSI-derived edema fraction, suggesting the presence of vasogenic edema in several WM regions (tapetum was chosen as a representative example, Fig. 1A). Significantly reduced DBSI-derived axial diffusivity suggested the presence of axonal injury at this early AD dementia stage (Fig. 1B). In comparison, the significantly increased DTI-derived axial diffusivity suggested the failure of DTI to detect axonal injury, probably due to the confounding effect of vasogenic edema (Fig. 1B, red arrow). DTI-/DBSI-derived radial diffusivity and FA increased (Fig. 1C) and decreased (Fig. 1D), respectively, in the early AD dementia group, suggesting demyelination and white matter abnormality. However, DBSI-derived radial diffusivity and FAwere not affected by vasogenic edema contamination and therefore reflected the white matter integrity with higher specificity and accuracy. Conclusions: Our data suggest that DBSI can separately quantify WM structural abnormalities and vasogenic edema in early symptomatic AD. In comparison, conventional DTI cannot separate the effect off WM abnormality from edema, limiting its usage in accurately characterizing the complicated pathological substrates in AD.